Project Summary We are currently investigating the use of human mesenchymal stromal cells (MSCs) for tissue repair by injecting MSCs into the damaged organ. Recent discoveries indicate that many of the therapeutic benefits of MSCs can be attributed to secretion of various biomolecules which can be secreted via exosomes, small membrane vesicles of endocytic origin. Most cell types secrete exosomes which contain proteins, DNA, mRNA, and microRNA, all of which which are thought to play a role in cell-cell communication. While previous research efforts largely focused on the characterization of proteins found in exosomes, our current research focuses on exosomal RNAs. Increasing evidence suggests that exosome formation and release are regulated by the autophagy pathway, a homeostatic quality control pathway that recycles proteins and organelles via recognition, sequestration, and lysosomal degradation. Conditions that stimulate autophagy pathway can inhibit exosome release, but at the same time pharmacological inhibitors of autophagy enhance the release of exosomes. For our study, we propose utilizing a subtype of MSCs called ?marrow-isolated adult multilineage inducible? (MIAMI) cells due to their ease of isolation from bone marrow, differentiation capacity, their immunomodulatory and tissue repair capacities, and ability to secrete various chemokines/growth factors. We hypothesize that autophagy mediates release of exosomes from MIAMI cells, regulates their RNA content and their immunomodulatory capacity. To stimulate MIAMI cells, we will expose them to inflammatory response stimulator, IFN?, while simultaneously applying pharmacologic stimulators or inhibitors of autophagy. Subsequently, we will isolate and characterize MIAMI cell-derived exosomes by using NanoSight, electron microscopy and immunoblotting to characterize and compare exosomes size distributions, exosome yield and markers (CD9, CD63 and CD81) (Aim 1). We will then determine how modulation of autophagy regulates exosomal RNA content by identifying and validating long (more than 200 nucleotides) and short (less than 200 nucleotides) RNAs in MIAMI cells-derived exosomes (Aim 2). Lastly, we will evaluate immunoregulatory properties of MIAMI cells upon modulation of autophagy using MIAMI cell-T cell co-cultures and subsequent flow cytometry analysis to assess T cell markers of proliferation and cytokine secretion (Aim 3). Results of this mechanistic study will increase our understanding of the role that autophagy plays in regulating RNA content in exosomes and it will also reveal whether targeting autophagy could be used to manipulate RNA content and subsequently immunomodulation. Ultimately, such knowledge is anticipated to foster further development of cell therapies for tissue regeneration. 1